Preparation of cobalamins



United States This application is a continuation-in-part of application Serial No. 642,087, led February 25, 1957, and now abandoned.

This invention relates to an improved process for preparing physiologically active cobalamins.

Prior to this invention, it was known that physiologically active cobalamins could be prepared biosynthetical* ly by culturing certain microorganisms in a cobalt-containing nutrient medium. When the nourmal culture medium components failed to provide adequate utilizable cobalt, additional cobalt, generally in the form of a salt, had to be added to the nutrient medium either before or during the incubation period. Since cobalt is known to have high toxicity to microorganisms, care had to be taken in such a process to assure that the concentration of cobalt in the medium was below its toxic level s o as to preventvany adverse effect ou the growth and metabolism of the microorganism. In order to prevent this possibility, therefore, the cobalt was added to the medium in low concentration, which often resulted in an insuiciency of cobalt and, hence, ya failure to fully convert all utilizable metabolites to the desired cobalamin.

It is the object of this invention, therefore,lto provide an improved process for the preparation of physiologically active cobalarnins which is more ecient and less critical than those previously known.

These objects are achieved by the process of this invention, which essentially comprises culturing a vitamin B12-producing microorganism in a cobalt deficient nutrient medium, separating the cells from the medium, treating the separated cells with a utilizable source of cobalt, aerating the resulting cellular suspension and recovering the resulting physiologically active cobalamin. [By cobalt deficient is meant, of course, a cobalt content (if any) insucient for maximum cobalarnin production.]

Among the microorganisms which may be employed in the practice of this invention are those known to produce Vitamin B12. known to form vitamin B12 Without added precursor, such as Streptomyces grseus, Streptomyces aureofaciens, Streptomyces albidoflavus, Streptomyces, antibioticus, Streptomyces colombiensis, Streptomyces fradiae, Streptomyces roseochromogenus, Streptomyces olvaceus, Propz'ombacterum freudenreichz', Aerobacter areogenes, Ashbya gossypii, Mycobacterium phlei, Mycobacterium smegmatis and Mycobacterium tuberculosis, as Well as microorganisms which are known to form vitamin B12 These include microorganisms which are the cobalamin and may be exemplified by 5,6-dimethylbenzimidazole, which is a precursor for vitamin Bm] Furthermore, the use of an added precursor-requiring microorganism afford-s a process whereby unnatural physiologically active cobalamins can be prepared, this modication of process being more fully detailed herein after.

The nutrient media useful inthe first step of the process of this invention include the usual sources of assimilable carbon and nitrogen. As sources of assimilable carbon', there may be used: (l) carbohydrates, such as glucose, fructose, sucrose, maltose, dextrins and soluble starches; (2) substances containing carbohydrates, such as corn steep liquor and grain mashes; (3) polyhydric alcohols, such as glycerol; (4) fats, such as lard oil,-soybean oil,

linseed oil, cottonseed oil, peanut oil, coconut oil, corn oil, castor oil, sesame oil, palm oil, mu-tton tallow, sperm oil, olive oil, tristearin,'triolein and tripalmitimand v(5) fatty acids having more than 14 carbon atoms, such as stearic acid, palmitic acid, oleic acid, linoleic acid and myristic acid.

Sources of available nitrogen include: (l) organic nitrogen compounds, such as proteinaceous materials, e.g. soybean meal, iish meal, casein, whey or whey concentrates, yeast, amino acids and liver cake; and (2)v inorganic compounds, such as nitrates or ammonium compounds. I

The cobalt-deficient nutrient media may, of course, contain any of the additional components usually found in such solutionsyamong these additional components are antifoarn agents (eg, lard oil, octadecanol, etc.), metallic cations, such as potassium, calcium, magnesium and iron (which may be present in the crude materials used in the nutrient medium), and phosphates (which may be added as inorganic phosphate).

The fermentation process may be carried out at temperatures from about 20 C. to about 40C. If the microorganism is one Which grows under aerobic conditions, a source of oxygen or air should also be present. This aeration can be accomplished by bubbling air (or oxygen) through the medium during the fermentation or by agitating the medium, thereby exposing a large surface thereof to the atmosphere. If the microorganism is anaerobic in nature, of course, this aeration step must then be omitted.

After a suiiicient incubation time (about one to ten days), the medium is separated into its liquid and solid only in a nutrient medium in which has been included a isrn in the biosynethesis of the base ofthe nucleotide of A components, as by filtration or centrifugation; and `the cells are suspended in water (preferably distilled water). This suspension can then, if desired, be again separated into its solid and liquid portions and the solid component resuspended in Water. The optimal pH of the finally adjusted suspension is one in the range of about 5 to about 8.

To the suspension is added at least the stoichiometrically required amount of cobalt to yield the desired oo balamin. Unlike priorly known-processes, an excess of cobalt need not be avoided, so that the upper limit of permissible cobalt is not critical. This cobalt is added in the form of any utilizable source of cobalt, preferably in the form of an inorganic cobalt salt (e.g., cobalt nitrate, cobalt chloride). The temperature of the suspension during this addition is not critical and can be anywhere in the range of about- 10 C. to about 40 C. but preferably, for ease inhandling, is atambient temperature.

The resulting cellular suspension is then aerated in order to produce the desired physiologically active cobalamin. The aeration can be accomplished in any suitable manner, as for example, by bubbling air (or oxygen) through the suspension or by agitatin-g the suspension, thus exposing a larger surface thereof to the atmosphere.

It has ybeen found that this aeration step is essential for the production of the cobalamine, no signiiicant activity being observed prior thereto.

The thus-treated cellular suspension is then heated (e.g., `to a temperature in the range of about 70 C. to about 100 C.), or lacidiiied (as disclosed in Patent No.

2,656,300 of McCormack et al., granted October 20,v

1953), or disrupted -by treatment with supersonic energy to release the cobalamin from the cells, and then ltered or centrifuged, and the cobalamin recovered from the liquid'portion. As an alternative procedure, the cells are first separated from the liquid and then lysed, as by treatment with a solvent such as acetone or n-propancl.

The recovery step can be further modilied by concentrating the cellularsuspension, after treatment with cobalt and aeration and before rele-ase of the cobalamin from the cells, as by centrifuging; the supernatant fluid (which contains only a smal-1 amount of the cobal'amin) is discarded, and the concentrated cellular suspension is then heated, acidiedor disrupted as described hereinbefore to release the cobalamin from the cel-ls.

Furthermore, lif an animal feed supplement is desired, the treated cellular suspension may be dried and incorporated directly into animal feeds.

If an added precursor-requiring rncroorganism is employed, the precursor can be added either directly to the nutrient medium, or to the cell suspension. The nature of the precursor employed depends on the physiological-ly active cobalamin desired. Thus, if vitamin B12 is desired, 5,6-dimethylbenzimidazole or lanother vitamin Bmprecursor, such as 2,3dimethyl5,6diamino benzene, 2,S-dirtro-5,6dimethylbenzene Vor 2,3-dmethyl-4-amino5-nitrobenzene, may be used. If an unnatural vitamin B12 which possesses vitamin B12-like activity is desired, a precursor for the particular cobalamin vcan be used. Thus, as summarized in the following table, the nature of the resulting cobalamin will depend on the precursor chosen; and since this choice is independent uof the first fermentation step, this aspect cf the of vthis invention affords a method whereby 'a variety of cohalamins can be produced from the product of a single fermentation procedure. In this Itable, the 'symbol Y is that in accompanying drawing:

VProduct (Y-cobalamtns) Precursor Name Y is 5,6-Dimethylbenzlmidazole.

A methylbenzene.

2,3-Dimethyl-4- amino-S-nitrobenzene.

nemmerhyi.

benzimidazolecobalamin. G-CHs 1,2-Diaminobenzene.

lgQ-D initrobenzene.

1-Arnno-2-nitrobenzene.

Benzmidazolecobalamiu.

-Triuor'omethylbenzimidazole. eobalamin.

4-Bromo--methoxybenzimidazole.y

Brorno--methoxyphenylene-dia- B11112452.

'Hummelo-meth- Oxy-benzimidazole cobalamin.

Product {Y-cobalamins) Precursor Name Yis- Quiuazoline Quiuazolineeobalamin.

il nam-quirom- -aeDuiydro-r limone. oxoquinazol line-cobalamin. N l

.t L 2,4-Dicnioroquin- 2,4Dich1omo1-// azoliue. quinazoline. I

cobnlamin. N

l Cl

NO2 l l N\ 4-Chloro-8-ntro- 4-Chloro-8mitrof quiua'zoline. quinazolinecohalamin. N

Y l l. :tornai-Quale naal-remm- 'o-/ zeiinedione. amen-dior@- l quina oline- N cobalamiu.

'-lkmrino-llmethoxy- S-lrrniuo-t-methqulnazoline. oxy-quinazoline-cobalamin.

2-B/Iethy1-t-methyl- Z-Methyl-4- thioquinazoliue. methyl-thi@ quiuazoliuecobalumin.

Pltieuazine Phenazinecobalamin.

'2Nitr'ophenaz`ine.--; @Nitrophenazineeobalamn.

2-Aminophenszine.. Z-Aminophen- 'ezine-cobalamin.

1`Ami11o2hydroxyla'mino-Z-hyphenazin'e. droxy-phem azine-coba1- amu.

Product (Y-cobalamns) Precursor Name Y ls I N H2 C O C H2 N 1-Acetamdo-3- 1-Acctemido-3- rnethoyyphem methoxyphennzine. ezine-cobalarnin. -O C H3 N Z-Hydroxyphen- Z-Hydroxyphen- -OH azine. azure-cobalamm.

\N t s C C-f-CHs fMcthylbenzimida- 5-Methylbenzi- OH zole. milglrzola /C\ /C co a amm. N C H ,lr l. Quinoxaline Quinoxalinej cobalamin. L

ln the drawing X represents an anion, for example, a hydroxy radical or the anion of an acid (preferably a pharmacologically acceptable acid). Examples of suitable anions are the anions of mineral acids (e.g., chloride, bromide, sulfate, nitrite and nitrate), cyanide, cyanate, etc. If no utilizable anion is present either in the fermentation medium or added with the cobalt in the second step, a hydroxy-cobalamin is initially formed (X=OH). If, however, the cell suspension to which cobalt has been added contains cyanide ion, a cyanocobalamn is recovered as the product (X=CN). Furthermore, if a particular salt is desired, the hydroxy-cobalamin can be converted to the salt by treatment with an acid. Thus, a hydroxy-cobalarnin, upon treatment with hyd-rochloric acid, yields the chloride (X=Cl), or with hydrogen cyanide (or potassium cyanide in an acidic medium), yields the cyanide (X=CN) To show the homogenity and activity of the cyanocobalamins formed in the examples of this invention, the following tests were conducted. For these tests the cyanocobalamin was dissolved in water to give `a concentration of about 100 micrograms of cyanocobalamin per ml. of water:

TEST I The solution of the cyanocobalamin is dried on a filter paper strip of Whatman 3 MM paper in parallel with samples of 5,6- diniethylbenzimidaZoie-cyanocobalamin, adenine-cyanocobalamin, 2 methyl-adenine-cyanocobalamin and Fords Factor B [Ford et al., Biochem, Jour., 59, 86 (1955)]. The sheet is placed in an ionophoresis apparatus [similar to that described by Holdswotth in Nature, 171, 148 (1953)], and the paper is impregnated with a solution of 0.5 N acetic acid containing 0.02% KCN (w./v,). A potential of about 280 volts is applied for about 17 hours. The sheet is removed and dried. When dry (and free from odor of acetic acid), it is applied for minutes to the surface of an agar plate seeded with -a suspension of a vitamin B12-requiring strain of Escherichia coli (ATCC 11105). [The agar medium contains (grams/liter): sucrose, g.; citric acid, 1.2 g.; (NH4)2HPO4, 0.4 g.; KCl, 0.08 g.; MgClz'I-lzO, 0.418 g.; MnCl2-4H2O, 0.036 g.; FeCl3-6H2O, 0.023 g.; ZnClZ, 0.021 g.; CoCl2-6H2O, 0.04 g.; agar, 15 g.; triphenyl tetrazolium chloride, 1 -g.] After 18 hours incubation at 37 C., the agar plate is observed. The positions ofl zones Vmethylbenzimidazole-cyanocobalamin as standard.

tained'at 150) fare added to each placed on a reciprocating shaker of growth of bacteria (noted as red zones'due to the reduction of the tetrazolium dye to the colored formazan) are noted in relation to the location on the paper strip where the samples are applied. The lresults obtained are recorded inthe examples.

TEST II An aliquot of the solution is applied to a spot about 3 inches from the end of a strip of Whatmau No. 1 tilter paper parallel to spots of known cobalamins. The chromatogram is developed by the descending method using a solvent mixture containing: 77 m1. of sec-butanol, 23 ml. of water, 0.25 ml. of KCN solution (5 gms./ 100 m1.) and mg. of KClO4 for 24 hours (at 25 C.). The strip is dried and applied to the seeded agar plate as in Test I. After incubation, the zones of growth, representing the presence of vitamins of the B12 group (measured with reference to the movement of 5,6-dimethylbenzimidazole-cyanoc0balamin) are determined.

TEST III (a) An aliquot of the solution is applied to a spot about 3 inches from the end of a strip of Whatrn-an No. 4 filter paper parallel to spots of known cobalamins. The chromatognam is developed by the descending method using a solvent mixture containing: sec-butanol, 100 ml.; water, 50 m1.; KCN [5 solution (w./v.)], 0.25 ml.; and NH4OH (concentrated), 1.0 ml. After 17 hours development (at 35 C.), the strips Iare dried and plated on seeded agar plates as in Test I. Zones of growth are determined.

(b) Same as Test Illa with 1.0 ml. of glacial acetic acid substituted for the ammonium hydroxide.

TEST IV vmacopia (15th edition). A value is determined.

TEST V An aliquot is -assayed by the method of Ford and Porter [Brit I. Nutrition, 7, 326 (1953)], using the growthresponse of Ochromonas mnllmmenss and 5,6-di- A value is obtained.

The following examples illustrate the invention (all temperatures being in centigrade):

Example 1 VITAMIN Bn A medium containing 2O grams autolyzed yeast, 30 grams glucose and 1 liter oi' tap Water is prepared, and k50G-ml. aliquots are placed in l-liter Erlenmeyer asks. The asks are plugged with non-absorbent cotton and autoclaved at 121 C. for 30 minutes. Approximately 10 grams of powdered calcium carbonate (previously sterilized by heating for at least 3 hours in an oven mainask. When the liquid has cooled to 30, the flasks are inoculated with 10 ml. of 2-day-old culture of Propionbacterz'um arabnosum ATCC 4965 (American Type Culture Collection, Washington, DC.) grown on this medium. The flasks are then (-1 inch cycles per minute), located in a constant temperature room maintained at 30. After 48 hours to 74 hours of incubation, the cells, Vdebris and calcium carbonate in the ask are collected by centrifugation. The collected solids are resuspended in a volume of distilled water equal to the original,

.shaken on a reciprocating shaker (280-1 inch cycles The pH of the suspension is about pH 6.2. Approximately 200 mg. of 5,6-,dimethylbenzimidazole and cobalt 'nitrate [in sucient quantity to give a concentration of 1 ing. of cobalt per liter, eg., 5 mg. of Co(NO3)2r6H2O] are added to one of the series of asks, and the flasks are placed on a reciprocating shaker (120--1 inch cycles per minute), located in a room maintained at 30. After 20 hours agitation, the pH of the suspension rises to about 6.7. The contents of the flasks are heated at 85 to 90 C. for 20 minutes (in a boiling water bath), and the suspension is centrifuged. Approximately 0.5 ml. of an aqueous solution of KCN (5 g./ 100 ml.) is added to 20 ml. of the supernatant liquid. An aliquot of this supernatant liquid (hereinafter referred to as the supernatant liquid) is analyzed for the presence of substances stimulating the growth of Lactobacillus Zeclzmannii (ATCC 7830), using as standard 5,6dimethylbenzimdazole cyanocobalamin and the method in the U.S. Pharmacopia (fifteenth edition) (Test IV). A value of about 0.47 mg. per liter is obtained. Another aliquot approximately ml. in volume is shaken with 10 ml. of a mixture of phenol and benzene (70 parts 88% phenol-30 parts benzene). The mixture is centrifuged, and the upper layer (ca. 8 ml.) is transferred to a test tube. An equal volume of n-butanol is added to this phenol-benzene extract, and the solution is shaken brieiiy. Five m1. of Water is then added, the mixture shaken on a reciprocating shaker for 10 minutes and then centrifuged. The bottom aqueous layer (hereinafter called the aqueous concentrate) is removed land analyzed by the following tests:

TEST I In this test the major cobalarnin present in the aqueous concentrate has a mobility equal -to that of 5,6-dimethylbenzimidazole-eyanocobalarnin. VThere is also some adenine-cyanocobalamin present. A sample of the aqueous concentrate from a cell-suspension to which cobalt has been added but which has not been supplemented with the 5,6-dimethylbenzimidazole shows only the presence of adenine-cyanocobalamin.

TEST II In this test the maior cobalamin present in the laqueous concentrate has a mobility equal to that of 5,6-dimethylbenzimidazole-cyanocobalamin. There is also a `cobalarru'n present which has a mobility about 0.35 that of the 5,6dimethylbenzimidazole-cyanocobalamin or Lequal to that of adenine-eyanocobalamin.

TEST Illa In this test the major cobalamin present in the aqueous concentrate has a mobility equal to that of 5,6-dirnethylbenzimidazole-cyanocobalamin. There is Aalso a cobalamin present with a mobility about 0.6 that of the 5,6- dimethylbenzimidazclecy-anocobalamin.

rnsr nib the supernatant liquid is assayed growth of Another aliquot of for the presence of substances stimulating the Ochromonas malhamensz's, using the method of Ford and Porter [Brit J. Nutrition, "7, 326 (1953)] with 5,6-dimethylbenzimidazole-eyanocobalamin as standard. A value of `about 0.4 mg. per liter is obtained. When an -aliquotof the supernatant liquid from an unsupplemented 'cell-suspension is assayed, a value of (0.01 mg. per liter is obtained.

Other. substances which may be used instead of the 5,6? dimethylbenzimidazole in the procedure of Example 1 include: V2,3-dimethyl-5,6-diarninobenzene; 2,3-dinitro5,6 dimethylbenzene', or 2,3dimethyl-4-amino-Smitrobenzene. The cell-suspension may be shaken in Ian atmosphere of nitrogen instead of air without atecting the biosynthesis of the cobalamin.

Example 2 VITAMIN B12 The same procedure as used in Example 1 is used with a culture of Propz'onibaterium pentosaceum ATCC 4875 instead of the P. arabinosum. The supernatant liquid contains about 0.3 mg. per liter of 5,6-dimethylbenzimidazole-cyanocobalamin, as measured by either the L. lechmmmii bioassay (Test IV) or the 0. malham'enss bioassay (Test V). The aqueous concentrate contains 5,6 dimethylbenzimidazole cyanocobalamin, as determined by the methods of Tests I through IIIb. When the cobalt salt is not added to the cell suspensions, no cobalamin is formed, while omission of the 5,6-dimetbylbenzimidazole results in the formation of only adeninecyanocobalamin as `determined by the procedures of Tests 1 through IV and Kthe lack of activity in the 0. mal- Izamensis bioassay (Test V).

Example 3 VITAMIN Bia The procedure of Example 1 is used with the replacement of the P. arabnsum culture Vwith P. freudenreiclziz' ATCC 6207 and omission of the 5,6-dimethylbenzimidazole. Examination of the aqueous concentrate shows the presence of 5,6-dimethylbenzimidazole-cyanocobalamin, as determined by the procedures of Tests I to IV. When cobalt is omitted from the mixture, no cobalamin is detected in the supernatant liquid. 'The bioassay of the supernatant liquid is about 0.4 mg. per liter, as measured -by the L. lechmanni (Test IV) and O. nmlhamensisV (Test V) bioassays (using 5,6-dimefhylbenzimidazolecyanccobalamin as standard).

Example 4 BENZIMIDAZOLE-CYANOCOBALAMIN The procedure described in Example 1 is used with the replacement of the 5 6-dimethylbenzimidazo1e with benzimidazole. The bioassay of the supernatant liquid shows about 0.5 mg. per liter of activity by the L. lechmannii assay (Test 1V.) 5,6 dimethylbenzimidazole cyanocobalamin being used as standard.

The same results are obtained when o-phenylene diamine or o-dinitrobenzene is substituted for the benzimidazole.

Example 5 BENZIMIDAZOLE-CYANOCOBALAMIN The procedure described in Example 2 is used with the replacement of the 5,6-dimethylbenzimidazole with benzimidazole. The bioassay of the supernatant liquid shows about 0.5 mg. per liter of activity by the L. lechmannii assay (Test 1V). Analysis of the aqueous concentrate shows the presence of benzimidazole-cyanocobalamin, as described in Example 4.

Example 6 5-TRIFLUOROMETHYLBENZIMEAZOLECYANG COBALAMIN The procedure used in Example 1 is used with the replacement of the 5,6-dimethy1benzimidazole with 0th,@- triuoromethyl-Z-nitro-p-toluidine. The bioassay of the supernatant liquid `shows about 0.4 mg. per liter-of activity by the L. leichmanm'i assay (Test IV), 56-dimethy1benzimidazole-eyanocobalamin being used as a standard. Analysis of the aqueous concentrate by the procedure of Test I shows thepresence -of aneutral cobalamin (ionophoretically). Analysis of the aqueous concentrate by Test II-I shows a cobalamin with a mobility of about 0.95 that of the 5,G-dimethylbenzimidazole-cyanooobalamin, and the same result is obtained when analysis is made of the aqueous concentrate by the procedures of Tests IIIa and IIIb. When 3,4-diamino-a,,a-triuoromethyltoluene lis substituted forthe trilluoro-Z-nitro-p-toluidine, the same results are obtained.

Example 7 Example 8 -HETHYLBENZIMIDAZOLE-CYANOCOBALAMIN By replacing the ,-dimethylbenzirnidazole in Example l with S-methylbenzimidazole, there is obtained 5methyl benzimidaZole-cyanocobalamin.

Example 9 QUINOXALINE-CYANOCOBALAMIN The procedure used in Example 1 is used with the replacement of`5,G-dimethylbenzimidazole with quinoxaline hydrochloride to yield quinoxaline-cyanocobalamin.

Example 10 BENZOTRIAZOLE-CYANOCOBALAMIN The procedure of Example 1 is used with the replacement of 5,6-dimethylbenzimidazole with benzotriazole. The bioassay of the supernatant liquid is 0.9 mg. per liter as measured by the L. leichmanni method (Test IV) and 0.3 mg. per liter as measured by the O. malhamenss bioassay (Test V), using 5,6-dimethylbenzimidazolecyanocobalamin as a standard. Analysis of the aqueous concentrate by the procedure of Test I shows the presence of an ionophoretically neutral cobalamin. Analysis of the aqueous concentrate by the procedure of Test II shows the presence of a cobaiamin with a mobility of 1.05 that of 5,-dimethylbenzimidazole-cyanocobalamin, While when the procedure of Test IIIb is used, the new cobalamin has a mobility equal to that of 5,6dimethylbenzimidazole cyanocobalamin. When the procedure of Test IIIa is used, the, new cobalamin has a mobility of about 0.3 that of 5,6-dimethylbenzimidazole-cyanocobalamin.

Example 1l PHENOTHIAZINE-CYANOCOBALAM IN The procedure of Example 1 is used with the replacement of the 5,6-dimethylbenzimidazole with phenothiazine. The'supernatant liquid has an activity of about 0.4

mg. per liter as measured by the bioassay based on the growth response of L. Iechnzannz' (Test 1V) (with 5,6-di- 60 methylbenzimidazole-cyanocobalamin as standard). When the aqueous concentrate is examined by the procedure of Test I, an ionophoretically neutral substance is found. When the procedure of Test II is used, the new cobalamin has a mobility of about 0.46 that of 5,6-dimethylbenzimidazole-cyanocobalamin.

Example 12 PHENOTHInZINE-CYANOCOBALAMIN The procedure of Example 2 is used with the replacement of the 5,6-dimethylbenzimidazole with phenothiazine. The supernatant liquid has an activity of about 0.4 mg. per liter as measured by the bioassay based on the growth response of L. leclzmannz' (with 5,6-dimethylbenzimidazole-cyanocobalarnin as standard). The aque- 1&9 ous concentrate contains the same eobalamin described in Example 11.

Example 13 VITAMIN B1.'-

A medium containing 5 grams of meat extract, 10 grams peptone (Bacto) and 10 grams glucose is diluted to one liter with distilled Water. Seventy-five ml. aliquots are distributed into Z50-ml. Erlenmeyer flasks and autoclaved for 20 minutes at 126 C. When cooled to 30 C., the flasks are inoculated with Streptomyces aureofacens (NRRL 2209) and placed on a rotary shaker located in room maintained at 25 C. After 2 days, 1 ml. of the growth is used to inoculate the same medium in a second flask, and the inoculated ask is placed on the shaker. After 2 days incubation, the cells are collected by centrifugation and resuspended in distilled water. The centrifugation is repeated and the cells resuspended in a second volume of distilled water. (The dry Weight of the cells is about 4.5 mg. per ml. of suspension.) Aliquots of the suspension are distributed into llasks (20 ml. per 12S-ml. Erlenmeyer ask is a convenient volume), and cobalt nitrate solution is added to give a nal concentration of about 1 mg. of cobalt per liter. The asks are placed on a reciprocating shaker located in a room maintained at 30 C. for about 18 hours. At the end of this interval, the pH is adjusted to about 2.5 by the addition of 12 N sulfuric acid; and the suspension is shaken and then centrifuged (this releases the cobalamins). The supernatant liquid is neutralized to about pH 7.5 by the addition of solid NaI-1G03; 0.5 ml. of a 5% solution of KCN is added and assayed for cobalamin content using the bioassay based on the growth response of L. leichmanm'i (Test IV), with 5,6dimethylbenzimidazole-cyano- 'oobalamin as standard. Approximately 0.3 mg. perI liter are found. When the supernatant liquid is extracted by the phenol-benzene solvent, as described in Example 1, and the aqueous concentrate is prepared, the latter is found to contain 5,6-dimethylbenzimidazole-cyanocobalamin by the procedures of Tests I to III.

Example 14 VITAMIN B11,v

Example 15 VITAMIN Bw, Y

Y17.8 liters of a medium containing (per liter): glucose, 51 grams; autolyzed yeast, 34 grams; tap water, 1 liter; are placed in a stainless-steel fermentation unit of 38 liters capacity, heated at 121 C. for 30 minutes by injection of steam and cooled to 30 C. (final volume 28 liters). About 2,000 ml. of a slurry of CaCO3 (containling 600 g. of CaCOB), sterilized by autoclaving is then added, together with 1 liter of Propionibacterum freudenrechz'z' ATCC 6207 culture grown on this medium for 48 hours in asks shaken on a reciprocating shaker (-1 inch strokes per minute), located in a constant temperature room. Maintained at 30 C., the culture is allowed to grow in the medium under virtually anaerobic conditions while being agitated with a turbine mixer rotating at 87 r.p.m. After 18 hours incubation at 30 C., concentrated ammonium hydroxide is added to adjust the pH from 5.7 to 6.0. After a total of 42 hours incubation when the pH is 5.2, the fermented medium is passed through a Sharples Super Centrifuge. The collected solids are resuspended in 3 liters of distilled water, and the suspension is added to 17 liters of water in the stainless-steel fermentation unit. Approximately 200 mg. of cobalt nitrate hexahydrate are added and the cell suspen :menace C obalamin v content,2 mg./l.

Identity of coonlamn by Length ci incuba- Tests I to III tion period (hours) pH 5,t-Dimethylbenzirnidazolecyanocobalamin.

racism-ocio ence @wang l Before addition of the cobalt nitrate.

2 As determined by stimulation ofthe growth of Lactuhacllus leichmannu (Test 1V) as described in Example 1 with 5,6dnncthylbenzimid azolecynnoco'halamin as standard.

Example 16 VITAMIN Bn The procedure of Example is used with the addition of cobalt nitrate in suilicient quantity to give a concentration of 10Q mg. of cobalt per liter of medium instead of the lower level. Examination of the aqueous concentrates by the procedures of Tests I to III shows they presence of 5,6-dimethylbenzirnidazole-cyanocobalamin. The bioassay ofthe supernatant liquid is about 1.0 mg. per liter as measured by the L. leichmannii bioassay (Test IV).

The cyanocobalamins formed in each of the examples can be converted to the corresponding hydroxocobalamin derivatives by treatment of the former with hydrogenV in .the presence of platinum oxide in an aqueous medium. The hydroxocobalamins thus formed can then be converted to any desired salt by treatment with the appropriate .acid in an aqueous medium.

The Yvitamin B12 and other biologically active cobalamins formed in each of the examples can be Vused in lieu of otherwise-produced vitamin B12 in promoting growth of chicks. For this purpose, the cobalarnin-containing superuate may be merely dried, to provide a cobalamin concentrate; or the cobalamin may be recovered from the supernate or dried concentrate by use of conventional vitamin B12 purification expedients. The dosage employed (e.g., when added as a supplement to chick feeds) would Adepend on the potency of the concentrate, or potency of the isolated non-B12 cobalamin, relative to pure vitamin B12.

The invention may 'be yotherwise variously embodied within the scope of the appended claims.

What is claimed is:

l. A process for preparing a physiologically active cobalamin, which comprises culturing a vitamin B12- producing microorganism in a cobalt-deficient nutrient medium, separating the cells from the medium, treating the separated cells with a utilizable source of cobalt, Yaerating the thus-treated cells and recovering the resulting physiologically active cobalamn.

Cil

2. The process of claim 1, wherein the utilizable source of cobalt is an inorganic cobalt salt.

3. The process of claim l, wherein the microorganism is of the genus Propionibacterium.

4. The process of claim 1, wherein the microorganismo is Srreptomyces aureofacens.

5. The process of claim l, wherein the microonganism is Propz'onibacterum freudenreichi.

6. A process for preparing a physiologically active cobalarnin, which comprises culturing an added precursorrequiring vitamin B12-producing microorganism in a cobalt-decient nutrient medium, separating the cells from the medium, treating the separated cells with a utilizable source of cobalt, aerating the thus-treated cells and recovering the resulting physiologically active cobalamin.

7. The process of claim 6, wherein the nutrient rnedium is substantially free of precursor and a precursor is added to the separated cells.

8. The process of claim 6, wherein a precursor is added to the nutrient medium.

9. The process of claim 6, wherein the micrcorganism is of the genus Propionibacterium l0. The process of claim 9, wherein 5,6-dimethylbenzimidazole is added as a precursor and a 5,6-dimethylbenzimidazole-cobalamin is recovered.

1l. The process of claim 9, wherein 5-methylbenzimidazole is added as a precursor and 5methylbenzimidazolecobalamin is recovered.

12. The process of claim 9, wherein benzimidazole is added as a precursor and benzimidazole-cobalamin is recovered.

13. The process of claim 9 wherein the microonganism is Proponibacterum arabinosum.

14. The process of claim 9 wherein the microorganism is Proponr'bacterum pentosaceum.

References Cited in the file of this patent UNITED STATES PATENTS 2,530,416 Wolf et al Nov. 21, 1950 2,595,499 Wood et al. May 6, 1952 2,643,213 Hall June 23, 1953 2,650,896 McDaniel Sept. l, 1953 2,683,681 McCormick July 13, 1954 2,715,602 Hargrave Aug. 16,1955 2,796,383 Robinson June 18, 1957 2,809,148 Bernhauer et al Oct. 8, 1957 2,842,540 Perlman July 8, 1958 2,872,444 Perlman Feb. 3, 1959 2,893,988 Bernhauer et al Iuly 7, 1959 FOREIGN PATENTS 948,734 Germany Mar. S, 1956 OTHER REFERENCES Germany, A 19,703 lV a/-30h, Mar. 8, 1956. Darlren: The Botanical Review, vol.v 19, No. 2, February 1953,l pp. -130. 

1. A PROCESS FOR PREPARING A PHYSIOLOGICALLY ACTIVE COBALAMIN, WHICH COMPRISES CULTURING A VITAMIN B12 PRODUCING MICROORGANISM IN A COBALT-DEFICIENT NUTRIENT MEDIUM, SEPARATING THE CELLS FROM THE MEDIUM, TREATING THE SEPARATED CELLS WITH A UTILIZABLE SOURCE OF COBALT, 